Available data suggest that distinctly different groups of microorganisms may be etiologically linked to individual forms of gingivitis and periodontitis. Whether these organisms actually invade periodontal tissues has been an issue of considerable debate. Inflammatory periodontitis was traditionally assumed to result directly or indirectly by soluble bacterial products. However, new evidence suggests that bacteria actually do invade the periodontium in localized juvenile periodontitis, ANUG, and in advanced periodontitis. In order to successfully invade tissues, the bacteria must actively or passively move through the epithelium, basement membrane, and underlying connective tissue. Thus, the basement membrane represents a major barrier to invasion of the underlying tissues. The ability of microorganisms to adhere to basement membrane is a prerequisite for colonization and ultimate invasion. The goal of this study is to develop a model system to investigate bacterial invasion through basement membrane-like matrix in vitro. Specifically, we propose to use the HR-9 matrix to: (1) Investigate and characterize bacterial attachment to the basal lamina-like matrix; (2) Test for specific degradation of macromolecules composing the HR-9 matrix by selected oral bacteria (suspected periodontal pathogens). For this purpose, the HR-9 matrix, consisting of Type IV collagen, heparin sulfate proteoglycans, laminin, etc., will be used as an in vitro model since it closely resembles true in vivo basement membrane. Cultures of PF HR-9 cells (derived from an embroyonal carcinoma) will be grown to confluency and the cells will be lysed by hypotonic lysis. These prepared cell-free matrices will initially be used in an adherence assay employing 3H-labeled bacteria. For later invasion and degradation studies, the matrices will be radiolabeled with 3H-leucine, 35S-sulfate or 3H-proline. Reference stock strains, as well as fresh clinical isolates will be grown to late log in supplemented TSB containing 3H-thymidine, washed x3 and suspended in buffered KCl(pH 6.0). Radiolabeled cells (5 x 10million) will be incubated with HR-9 matrix for 90 min at 37 degrees C and washed x3 with buffered KCl. Adherent cells will by lysed with IN NaOH, 0.05% SDS, and then counted by scintillation spectroscopy. The specificity of attachment to the matrices and the mechanisms of bacterial binding will be approached in several ways: competition experiments with purified components and use of specific antibodies. The gross loss of radioactivity from the matrices in the presence and absence of bacteria will indicate degradation of specific molecules. The extent of bacterial mediated solubilization of matrix components will be quantitated and alterations in solubilized components will be sized by gel filtration and identified by SDS-PAGE. It is likely that this study will provide us with information on the role of bacterial invasion in the pathogenesis of periodontal diseases.